Phosphatidylinositol 3-Kinase (PI3K) Orchestrates Aspergillus fumigatus-Induced Eosinophil Activation Independently of Canonical Toll-Like Receptor (TLR)/C-Type-Lectin Receptor (CLR) Signaling.

Eosinophilia is associated with various persisting inflammatory diseases and often coincides with chronic fungal infections or fungal allergy as in the case of allergic bronchopulmonary aspergillosis (ABPA). Here, we show that intranasal administration of live Aspergillus fumigatus conidia causes fatal lung damage in eosinophilic interleukin-5 (IL-5)-transgenic mice. To further investigate the activation of eosinophils by A. fumigatus, we established a coculture system of mouse bone marrow-derived eosinophils (BMDE) with different A. fumigatus morphotypes and analyzed the secretion of cytokines, chemokines, and eicosanoids. A. fumigatus-stimulated BMDE upregulated expression of CD11b and downregulated CD62L and CCR3. They further secreted several proinflammatory mediators, including IL-4, IL-13, IL-18, macrophage inflammatory protein-1α (MIP-1α)/CC chemokine ligand 3 (CCL3), MIP-1ß/CCL4, and thromboxane. This effect required direct interaction and adherence between eosinophils and A. fumigatus, as A. fumigatus culture supernatants or A. fumigatus mutant strains with impaired adhesion elicited a rather poor eosinophil response. Unexpectedly, canonical Toll-like receptor (TLR) or C-type-lectin receptor (CLR) signaling was largely dispensable, as the absence of MYD88, TRIF, or caspase recruitment domain-containing protein 9 (CARD9) resulted in only minor alterations. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway in A. fumigatus-induced eosinophil activation. Correspondingly, we could show that phosphatidylinositol 3-kinase (PI3K) inhibitors successfully prevent A. fumigatus-induced eosinophil activation. The PI3K pathway in eosinophils may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders. IMPORTANCE Allergic bronchopulmonary aspergillosis (ABPA) is caused by the fungus Aspergillus fumigatus, afflicts about five million patients globally, and is still a noncurable disease. ABPA is associated with pronounced lung eosinophilia. Activated eosinophils enhance the inflammatory response not only by degranulation of toxic proteins but also by secretion of small effector molecules. Receptors and signaling pathways involved in activation of eosinophils by A. fumigatus are currently unknown. Here, we show that A. fumigatus-elicited activation of eosinophils requires direct cell-cell contact and results in modulation of cell surface markers and rapid secretion of cytokines, chemokines, and lipid mediators. Unexpectedly, this activation occurred independently of canonical Toll-like receptor or C-type lectin receptor signaling. However, transcriptome analysis indicated a role for the PI3K-AKT-mTOR pathway, and PI3K inhibitors successfully prevented A. fumigatus-induced eosinophil activation. The PI3K pathway may therefore serve as a potential drug target to interfere with undesired eosinophil activation in fungus-elicited eosinophilic disorders.

An investigation into biomarkers for the diagnosis of ABPA and aspergillus disease in cystic fibrosis.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease due to Aspergillus fumigatus (Af) which occurs in 10% of patients with cystic fibrosis (CF). ABPA is associated with increased morbidity and accelerated lung function decline; however, existing diagnostic criteria are nonspecific and diagnosis remains challenging. As ABPA is driven by Th2 inflammation, the aim of this study was to evaluate exhaled nitric oxide (FE NO ), eosinophilic cationic protein (ECP), peripheral eosinophil count, and bronchodilator response (BDR) in patients with CF. METHODS: A prospective observational cohort study of pediatric CF patients in a tertiary center. Patients had a clinical and serologic ABPA assessment, FENO , serum ECP, peripheral eosinophil count, and assessment of BDR. Patients were stratified into three groups; ABPA, Af sensitized (AFS), and non-ABPA non-Af-sensitized (non-AFS). RESULTS: A total of 62 patients were included in the study: 13% ABPA, 19% AFS, and 68% non-AFS. Mean FENO was higher in the ABPA group at 37.8 ppb compared to AFS 15.1 ppb (P = .05) and non-AFS 13.7 ppb (P = .04). Mean peripheral eosinophil count in ABPA group was also higher at 1000 cells/uL, compared to AFS 221 cells/uL (P = .03) and non-AFS 220 cells/uL (P = .03). Mean BDR in ABPA group was 13% compared to 5.5% in non-AFS (P = .01). Serum ECP was higher in patients with ABPA positive compared to the other groups, although this was not statistically significant. CONCLUSION: In children with cystic fibrosis, FENO and peripheral eosinophil counts are elevated in ABPA compared to those that are just sensitized to Aspergillus and may serve as useful diagnostic tests.

Allergic Bronchopulmonary Aspergillosis-A Luminal Hypereosinophilic Disease With Extracellular Trap Cell Death.

Allergic bronchopulmonary aspergillosis (ABPA) is characterized by an early allergic response and late-phase lung injury in response to repeated exposure to Aspergillus antigens, as a consequence of persistent fungal colonization of the airways. Here, we summarize the clinical and pathological features of ABPA, focusing on thick mucus plugging, a key observation in ABPA. Recent findings have indicated that luminal eosinophils undergo cytolytic extracellular trap cell death (ETosis) and release filamentous chromatin fibers (extracellular traps, ETs) by direct interaction with Aspergillus fumigatus. Production of ETs is considered to be an innate immune response against non-phagocytable pathogens using a "trap and kill" mechanism, although eosinophil ETs do not promote A. fumigatus damage or killing. Compared with neutrophils, eosinophil ETs are composed of stable and condensed chromatin fibers and thus might contribute to the higher viscosity of eosinophilic mucus. The major fate of massively accumulated eosinophils in the airways is ETosis, which potentially induces the release of toxic granule proteins and damage-associated molecular patterns, epithelial damage, and further decreases mucus clearance. This new perspective on ABPA as a luminal hypereosinophilic disease with ETosis/ETs could provide a better understanding of airway mucus plugging and contribute to future therapeutic strategies for this challenging disease.

Blood basophil activation is a reliable biomarker of allergic bronchopulmonary aspergillosis in cystic fibrosis.

The diagnosis of cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA) is clinically challenging, due to the absence of an objective biological test. Since blood basophils play a major role in allergic responses, we hypothesised that changes in their surface activation pattern discriminate between CF patients with and without ABPA.We conducted a prospective longitudinal study (Stanford cohort) comparing basophil activation test CD203c levels by flow cytometry before and after activation with Aspergillus fumigatus allergen extract or recombinant Asp f1 in 20 CF patients with ABPA (CF-ABPA) and in two comparison groups: CF patients with A. fumigatus colonisation (AC) but without ABPA (CF-AC; n=13) and CF patients without either AC or ABPA (CF; n=12). Patients were tested every 6 months and when ill with pulmonary exacerbation. We also conducted cross-sectional validation in a separate patient set (Dublin cohort).Basophil CD203c surface expression reliably discriminated CF-ABPA from CF-AC and CF over time. Ex vivo stimulation with A. fumigatus extract or recombinant Asp f1 produced similar results within the Stanford (p<0.0001) and the Dublin cohorts. CF-ABPA patients were likelier to have elevated specific IgE to A. fumigatus and were less frequently co-infected with Staphylococcus aureus.Basophil CD203c upregulation is a suitable diagnostic and stable monitoring biomarker of ABPA in CF.

Long-Acting Beta Agonists Enhance Allergic Airway Disease.

Asthma is one of the most common of medical illnesses and is treated in part by drugs that activate the beta-2-adrenoceptor (ß2-AR) to dilate obstructed airways. Such drugs include long acting beta agonists (LABAs) that are paradoxically linked to excess asthma-related mortality. Here we show that LABAs such as salmeterol and structurally related ß2-AR drugs such as formoterol and carvedilol, but not short-acting agonists (SABAs) such as albuterol, promote exaggerated asthma-like allergic airway disease and enhanced airway constriction in mice. We demonstrate that salmeterol aberrantly promotes activation of the allergic disease-related transcription factor signal transducer and activator of transcription 6 (STAT6) in multiple mouse and human cells. A novel inhibitor of STAT6, PM-242H, inhibited initiation of allergic disease induced by airway fungal challenge, reversed established allergic airway disease in mice, and blocked salmeterol-dependent enhanced allergic airway disease. Thus, structurally related ß2-AR ligands aberrantly activate STAT6 and promote allergic airway disease. This untoward pharmacological property likely explains adverse outcomes observed with LABAs, which may be overcome by agents that antagonize STAT6.

Elevated levels of the neutrophil chemoattractant pro-platelet basic protein in macrophages from individuals with chronic and allergic aspergillosis.

BACKGROUND: Aspergillus fumigatus causes chronic cavitary pulmonary aspergillosis (CCPA) and allergic bronchopulmonary aspergillosis (ABPA) in overtly immunocompetent and atopic individuals, respectively. Disease mechanisms are poorly understood but may be related to increased neutrophil presence and activation. Pro-platelet basic protein (PPBP) is a potent neutrophil chemoattractant and activator whose expression is repressed by interleukin 10 (IL-10). METHODS: PPBP expression by monocyte-derived macrophages from patients with ABPA or CCPA and asthmatic and healthy controls (10 individuals per group) was analyzed using reverse-transcription polymerase chain reaction. PPBP and IL-10 protein levels in cell culture supernatants were measured by enzyme-linked immunosorbent assay. Two PPBP single-nucleotide polymorphisms (SNPs) were genotyped in 638 individuals. The gene was resequenced in 20 individuals. RESULTS: PPBP expression and protein levels were significantly increased in the ABPA (19.7-fold) and CCPA (27.7-fold) groups, compared with the control groups. PPBP SNPs were not associated with disease. IL-10 protein levels were significantly lower in the ABPA and CCPA groups, compared with the healthy group, suggesting that differences in PPBP levels may result from regulatory mechanisms. CONCLUSIONS: The results suggest a role for increased PPBP expression in ABPA and CCPA. Repression of PPBP expression may benefit some patients. Increased PPBP expression in ABPA and CCPA may be useful as a future diagnostic tool or possible target for novel therapeutics.

Pattern recognition pathways leading to a Th2 cytokine bias in allergic bronchopulmonary aspergillosis patients.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is characterised by an exaggerated Th2 response to Aspergillus fumigatus, but the immunological pathways responsible for this effect are unknown. OBJECTIVE: The aim of this study was to decipher the pattern recognition receptors (PRRs) and cytokines involved in the Aspergillus-specific Th2 response and to study Aspergillus-induced responses in healthy controls and ABPA patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were stimulated with heat-killed Aspergillus conidia, various other pathogens, or PRR ligands. PRRs and cytokine pathways were blocked with PRR-blocking reagents, anti-TNF (Etanercept or Adalimumab), IL-1Ra (Anakinra) or IFNγ (IFN-gamma). ELISA and FACS were used to analyse cytokine responses. RESULTS: Aspergillus was the only pathogen that stimulated the Th2 cytokines IL-5 and IL-13, while Gram-negative bacteria, Gram-positive bacteria, Candida albicans, chitin, ß-glucan or Toll-like receptor (TLR) ligands did not. Depletion of CD4(+) cells abolished IL-13 production. Blocking complement receptor 3 (CR3) significantly reduced IL-5 and IL-13, while blocking TLR2, TLR4 or dectin-1 had no effect. ABPA patients displayed increased Aspergillus-induced IL-5 and IL-13 and decreased IFNγ production compared with healthy controls. All biological agents tested showed the capability to inhibit Th2 responses, but also decreased Aspergillus-induced IFNγ. CONCLUSIONS AND CLINICAL RELEVANCE: Aspergillus conidia are unique in triggering Th2 responses in human PBMCs, through a CR3-dependent pathway. ABPA patients display a significantly increased Aspergillus-induced Th2/Th1 ratio that can be modulated by biologicals. These data provide a rationale to explore IFNγ therapy in ABPA as a corticosteroid-sparing treatment option, by dampening Th2 responses and supplementing the IFNγ deficiency at the same time.

Role of growth arrest-specific gene 6 in the development of fungal allergic airway disease in mice.

Growth arrest-specific gene (Gas)6 is a secreted vitamin K-dependent protein with pleiotropic effects via activation of receptor tyrosine kinase Tyro3, Axl, and Mertk receptors, but little is known about its role in allergic airway disease. We investigated the role of Gas6 in the development of fungal allergic airway disease in mice. The immune response was evaluated in Gas6-deficient (Gas6-/-) and wild-type (WT) mice and in recombinant Gas6-treated WT mice during Aspergillus fumigatus-induced allergic airway disease. Gas6 plasma levels were significantly elevated in adult clinical asthma of all severities compared with subjects without asthma. In a murine model of fungal allergic airway disease, increased protein expression of Axl and Mertk were observed in the lung. Airway hyperresponsiveness (AHR), whole lung Th2 cytokine levels, goblet cell metaplasia, and peribronchial fibrosis were ameliorated in Gas6-/- mice compared with WT mice with fungal allergic airway disease. Intranasal Gas6 administration into WT mice had a divergent effect on airway inflammation and AHR. Specifically, a total dose of 2 µg of exogenous Gas6 (i.e., low dose) significantly increased whole lung Th2 cytokine levels and subsequent AHR, whereas a total dose of 7 µg of exogenous Gas6 (i.e., high dose) significantly suppressed Th1 and Th2 cytokines and AHR compared with appropriate control groups. Mechanistically, Gas6 promoted Th2 activation via its highest affinity receptor Axl expressed by myeloid DCs. Intranasal administration of Gas6 consistently exacerbated airway remodeling compared with control WT groups. These results demonstrate that Gas6 enhances several features of fungal allergic airway disease.

Mesenchymal stromal cells mediate Aspergillus hyphal extract-induced allergic airway inflammation by inhibition of the Th17 signaling pathway.

Systemic administration of mesenchymal stromal cells (MSCs) suppresses airway inflammation and methacholine-induced airway hyper-responsiveness (AHR) in mouse models of T helper cell (Th) type 2-mediated eosinophilic allergic airway inflammation (AAI); however, the efficacy of MSCs in mouse models of severe Th17-mediated neutrophilic AAI has not yet been demonstrated. We assessed MSC effects in a mouse model of mixed Th2/Th17 AAI produced by mucosal exposure to Aspergillus fumigatus hyphal extract (AHE). Following sensitization produced by oropharyngeal AHE administration, systemic (tail vein) administration of syngeneic MSCs on the first day of challenge significantly reduced acute AHR predominantly through reduction of Th17-mediated airway inflammation. In parallel experiments, MSCs also mitigated AHR when administered during recurrent challenge 10 weeks after initial sensitization and challenge through reduction in systemic Th17-mediated inflammation. Investigation into potential mechanistic actions of MSCs in this model demonstrated that although T regulatory cells were increased in all AHE-treated mice, MSC administration did not alter T regulatory cell numbers in either the acute or recurrent model. Differential induction of interleukin-17a secretion was observed in ex vivo restimulation of mediastinal lymph node mixed-cell cytokine analyses. Although the mechanisms by which MSCs act to decrease inflammation and AHR in this model are not yet fully elucidated, decrease in Th17-mediated airway inflammation appears to play a significant role. These results provide a basis for further investigations of MSC administration as a potential therapeutic approach for severe refractory neutrophilic asthma.

Auto- and cross-reactivity to thioredoxin allergens in allergic bronchopulmonary aspergillosis.

BACKGROUND: Thioredoxins are cross-reactive allergens involved in the pathogenesis of atopic eczema and asthma. Cross-reactivity to human thioredoxin can contribute to the exacerbation of severe atopic diseases. METHODS: Human thioredoxin, Asp f28 and Asp f29, two thioredoxins of Aspergillus fumigatus, and thioredoxin of Malassezia sympodialis were cloned and produced as recombinant proteins. Allergenicity and cross-reactivity to thioredoxins in allergic bronchopulmonary aspergillosis patients were assessed by enzyme-linked immunosorbent assay (ELISA), inhibition ELISA, immunoblot analysis, proliferation assays and skin tests. Molecular homology modelling was used to identify conserved, surface-exposed amino acids potentially involved in immunoglobulin E (IgE)-binding. RESULTS: All thioredoxins, including the human enzyme, bind IgE from patients with allergic bronchopulmonary aspergillosis and induce allergen-specific proliferation in peripheral blood mononuclear cells and positive skin reactions in thioredoxin-sensitized patients. Inhibition experiments showed that the thioredoxins are cross-reactive indicating humoral immune responses based on molecular mimicry. To identify structural surface elements involved in cross-reactivity, the three-dimensional structures were modelled based on solved thioredoxin structures. Analysis of the molecular surfaces combined with sequence alignments allowed identification of conserved solvent exposed amino acids distantly located in the linear sequences which cluster to patches of continuous surface areas. The size of the surface areas conserved between human and fungal thioredoxins correlates well with the inhibitory potential of the molecules in inhibition ELISA indicating that the shared amino acids are involved in IgE-binding. CONCLUSIONS: Conserved, solvent exposed residues shared between different thioredoxins cluster to continuous surface regions potentially forming cross-reactive conformational B-cell epitopes responsible for IgE-mediated cross-reactivity and autoreactivity.

The role of CC chemokine receptor 6 in host defense in a model of invasive pulmonary aspergillosis.

RATIONALE: Invasive aspergillosis is a severe fungal infection afflicting immunocompromised patients, particularly patients with neutrophil defects. CCR6, a beta-chemokine receptor, mediates migration of dendritic cells (DCs) and several lymphocyte subsets to sites of epithelial inflammation, but its role in infections has not been examined extensively. OBJECTIVES: To test the hypothesis that CCR6-mediated leukocyte recruitment is necessary for effective host defense in neutropenic hosts with invasive pulmonary aspergillosis. METHODS: Neutropenic wild-type mice and mice with targeted deletion of CCR6 were infected with Aspergillus fumigatus. The host responses to the infection were compared in vivo and leukocyte responses to the fungus were examined in vitro. MEASUREMENTS AND MAIN RESULTS: In the context of infection, immature myeloid DCs were the major population of CCR6-expressing cells in the lungs. As compared with wild-type animals, CCR6-deficient mice developed a more severe infection when challenged with A. fumigatus conidia, as documented by a higher mortality rate and greater lung fungal burden. This was associated with reduced accumulation of DCs in the lungs. CCR6-deficient and wild-type DCs did not differ in their phagocytosis of conidia, cytokine response, or maturation in vitro. In adoptive transfer experiments, however, DCs from CCR6-deficient donors showed lesser accumulation in the lungs of infected mice as compared with wild-type cells, and transfer of wild-type, but not CCR6-deficient, DCs resulted in attenuated severity of infection in CCR6-deficient recipients. CONCLUSIONS: Taken together, these results implicate CCR6-mediated DC influx into the lung in the initial host defense in invasive aspergillosis.

A d-optimal designed population pharmacokinetic study of oral itraconazole in adult cystic fibrosis patients.

AIM: The primary objective of the study was to estimate the population pharmacokinetic parameters for itraconazole and hydroxy-itraconazole, in particular, the relative oral bioavailability of the capsule compared with solution in adult cystic fibrosis patients, in order to develop new dosing guidelines. A secondary objective was to evaluate the performance of a population optimal design. METHODS: The blood sampling times for the population study were optimized previously using POPT v.2.0. The design was based on the administration of solution and capsules to 30 patients in a cross-over study. Prior information suggested that itraconazole is generally well described by a two-compartment disposition model with either linear or saturable elimination. The pharmacokinetics of itraconazole and the metabolite were modelled simultaneously using NONMEM. Dosing schedules were simulated to assess their ability to achieve a trough target concentration of 0.5 mg ml(-1). RESULTS: Out of 241 blood samples, 94% were taken within the defined optimal sampling windows. A two-compartment model with first order absorption and elimination best described itraconazole kinetics, with first order metabolism to the hydroxy-metabolite. For itraconazole the absorption rate constants (between-subject variability) for capsule and solution were 0.0315 h(-1) (91.9%) and 0.125 h(-1) (106.3%), respectively, and the relative bioavailability of the capsule was 0.82 (62.3%) (confidence interval 0.36, 1.97), compared with the solution. There was no evidence of nonlinearity. Simulations from the final model showed that a dosing schedule of 500 mg twice daily for both formulations provided the highest chance of target success. CONCLUSION: The optimal design performed well and the pharmacokinetics of itraconazole and hydroxy-itraconazole were described adequately by the model. The relative bioavailability for itraconazole capsules was 82% compared with the solution.

Optimal design for model discrimination and parameter estimation for itraconazole population pharmacokinetics in cystic fibrosis patients.

Optimal sampling times are found for a study in which one of the primary purposes is to develop a model of the pharmacokinetics of itraconazole in patients with cystic fibrosis for both capsule and solution doses. The optimal design is expected to produce reliable estimates of population parameters for two different structural PK models. Data collected at these sampling times are also expected to provide the researchers with sufficient information to reasonably discriminate between the two competing structural models.

Immune regulation during allergic bronchopulmonary mycosis: lessons taught by two fungi.

Allergic bronchopulmonary mycosis (ABPM) is a devastating pulmonary disease that results from an aggressive allergic response to fungal colonization in the airways. Animal models using either fungal antigen or live infection reproduce most of the clinical features seen during ABPM in humans. Results from these studies have facilitated a detailed analysis of the key factors involved in the afferent as well as efferent phase of the disease. This review focuses on allergic bronchopulmonary disease caused by two different fungi (Aspergillus fumigatus and Cryptococcus neoformans): allergic bronchopulmonary aspergillosis and allergic bronchopulmonary cryptococcosis. Observations from both models underline the importance of initial innate immune responses and their translation into appropriate adaptive responses. In addition, data derived from knockout studies give emphasis to targeting cytokines and chemokines as a therapeutic strategy in the treatment of ABPM.

Profile of gene expression in a murine model of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) results from the interactions of the Aspergillus allergens and immune system of the patients. We studied the gene expression profile in a mouse model of ABPA. Of the 12,000 genes studied, 1,300 genes showed enhanced expression and represent chemokine, cytokine, growth factor, signal transduction, and transmembrane receptor genes as well as genes related to arginine metabolism.

Effect of amphotericin B treatment on kinetics of cytokines and parameters of fungal load in neutropenic rats with invasive pulmonary aspergillosis.

OBJECTIVE: The kinetics of various parameters of fungal load and cytokines were investigated, in order to acquire insight into the pathogenesis of invasive pulmonary aspergillosis (IPA) during antifungal treatment with amphotericin B. METHODS: Neutropenic rats with left-sided IPA received either treatment with amphotericin B or remained untreated. At 0, 4, 8, 16, 24, 48, 72 and 120 h after fungal inoculation, the rats were dissected. The size of the macroscopic pulmonary lesions, the number of cfu and amounts of chitin were determined in the infected left lung. Galactomannan concentrations were measured both in the left lung and serum. The cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, IL-4, IL-10, and the chemokines macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 were determined quantitatively by ELISA in the infected left lung, uninfected right lung and serum. RESULTS: Amphotericin B treatment of IPA resulted in changed aspect of pulmonary lesions and significantly reduced levels of left lung chitin (72 and 120 h), left lung galactomannan (72 and 120 h) and serum galactomannan (120 h), but not left lung cfu, compared with untreated infected rats. In addition, amphotericin B treatment resulted in a significant decrease in levels of left lung IL-6 (at 72 and 120 h), MIP-2 (at 120 h) and MCP-1 (at 120 h). No local or systemic increases in TNF-alpha, IL-1beta or IFN-gamma were observed during infection. CONCLUSION: It is concluded that treatment with amphotericin B results in decreased fungal load in the infected lung. This reduction in fungal load probably results in a decreased local inflammatory response, as measured by decreased levels of IL-6, MIP-2 and MCP-1 in the infected lung.

The late asthmatic response is linked with increased surface tension and reduced surfactant protein B in mice.

Pulmonary surfactant dysfunction may significantly contribute to small airway obstruction during the asthmatic response, but neither its exact role nor its regulation is clear. Surfactant function and composition was studied in an Aspergillus fumigatus (Af)-induced late-phase allergic airway response in sensitized BALB/c mice. The peak of Af-induced airway hyperresponsiveness in sensitized and challenged mice 24 h after allergen provocation coincided with a significant fall in surface activity of the pulmonary surfactant. The underlying changes included time-dependent elaboration of eotaxin and IL-5 followed by eosinophil influx into the airways. The height of airway inflammation and hyperresponsiveness was preceded by release of IL-4 and marked reductions in surfactant protein (SP)-B, a hydrophobic surfactant protein responsible for maintaining low surface tension of the lining fluid of distal air spaces. Furthermore, intratracheal administration of IL-4 significantly inhibited SP-B, indicating a regulatory role of this cytokine in the surfactant biophysical changes. Thus surfactant dysfunction induced by an IL-4-driven SP-B deficiency after allergen provocation may be an important part of the late asthmatic airway response.

Gamma-glutamyl leukotrienase, a novel endothelial membrane protein, is specifically responsible for leukotriene D(4) formation in vivo.

The metabolism of cysteinyl leukotrienes in vivo and the pathophysiological effects of individual cysteinyl leukotrienes are primarily unknown. Recently we identified an additional member of the gamma-glutamyl transpeptidase (GGT) family, gamma-glutamyl leukotrienase (GGL), and developed mice deficient in this enzyme. Here we show that in vivo GGL, and not GGT as previously believed, is primarily responsible for conversion of leukotriene C(4) to leukotriene D(4), the most potent of the cysteinyl leukotrienes and the immediate precursor of leukotriene E(4). GGL is a glycoprotein consisting of two polypeptide chains encoded by one gene and is attached at the amino terminus of the heavy chain to endothelial cell membranes. In mice it localizes to capillaries and sinusoids in most organs and in lung to larger vessels as well. In contrast to wild-type and GGT-deficient mice, GGL-deficient mice do not form leukotriene D(4) in vivo either in blood when exogenous leukotriene C(4) is administered intravenously or in bronchoalveolar lavage fluid of Aspergillus fumigatus extract-induced experimental asthma. Further, GGL-deficient mice show leukotriene C(4) accumulation and significantly more airway hyperreponsiveness than wild-type mice in the experimental asthma, and induction of asthma results in increased GGL protein levels and enzymatic activity. Thus GGL plays an important role in leukotriene D(4) synthesis in vivo and in inflammatory processes.

IgE down regulation and cytokine induction by Aspergillus antigens in human allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA), occurring primarily in patients with asthma or cystic fibrosis (CF), is a hypersensitivity reaction to Aspergillus fumigatus (Af), and is characterized by increased serum IgE levels and peripheral blood and pulmonary eosinophilia. We evaluated the IgE and cytokine profile in ABPA through enzyme-linked immunosorbent assay (ELISA), and evaluated eosinophil activity with the eosinophil peroxidase (EPO) assay. IgE and cytokines were measured in supernatants from cultures of peripheral blood mononuclear cells (PBMC) from three subject groups: ABPA patients, patients with asthma, and healthy individuals. All cultures for the three subject groups were studied in the presence and absence of two purified Af antigens (the 35-kD antigen and heat shock protein 1). We found that increased in vitro levels of IgE in unstimulated PBMC culture supernatants correlated significantly with serum IgE concentrations in ABPA patients. We measured a decrease in IgE levels of up to 75% of baseline values in supernatants from PBMC cultured with Af antigens. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) concentrations in cultures with Af were increased in ABPA, whereas concentrations of IL-4 did not differ in the three subject groups. An inverse relation was noted between the changes in IgE and IFN-gamma measured in 4 of 5 ABPA patients. The PBMC supernatants also promoted EPO activity in purified eosinophils from ABPA patients, and to a lesser extent in purified eosinophils from healthy subjects. These results show that the 35-kD antigen and HSP1 from Af downregulate IgE in vitro but are capable of inducing eosinophilia in ABPA. Further studies could result in the characterization of epitopes leading to these disparate effects. An identification of the IgE-down-regulating epitopes in Af antigens might have therapeutic significance.